Enrichment Methods for Murine Liver Non-Parenchymal Cells ...

Citation: Medina-Montano, C.;

Cacicedo, M.L.; Svensson, M.;

Limeres, M.J.; Zeyn, Y.;

Chaves-Giraldo, J.E.; Röhrig, N.;

Grabbe, S.; Gehring, S.; Bros, M.

Enrichment Methods for Murine

Liver Non-Parenchymal Cells

Differentially Affect Their

Immunophenotype and

Responsiveness towards Stimulation.

Int. J. Mol. Sci. 2022, 23, 6543.

https://doi.org/10.3390/

ijms23126543

Academic Editor: Jin-Kyu Park

Received: 20 May 2022

Accepted: 9 June 2022

Published: 11 June 2022

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International Journal of

Molecular Sciences

Communication

Enrichment Methods for Murine Liver Non-Parenchymal CellsDifferentially Affect Their Immunophenotype andResponsiveness towards StimulationCarolina Medina-Montano 1,†, Maximiliano Luis Cacicedo 2,† , Malin Svensson 2, Maria Jose Limeres 2,Yanira Zeyn 1 , Jean Emiro Chaves-Giraldo 1, Nadine Röhrig 1 , Stephan Grabbe 1 , Stephan Gehring 2,†

and Matthias Bros 1,*,†

1 Department of Dermatology, University Medical Center of the Johannes Gutenberg University Mainz,Langenbeckstraße 1, 55131 Mainz, Germany; [email protected] (C.M.-M.);[email protected] (Y.Z.); [email protected] (J.E.C.-G.); [email protected] (N.R.);[email protected] (S.G.)

2 Children’s Hospital, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1,55131 Mainz, Germany; [email protected] (M.L.C.); [email protected] (M.S.);[email protected] (M.J.L.); [email protected] (S.G.)

* Correspondence: [email protected]† These authors contributed equally to this work.

Abstract: Hepatocytes comprise the majority of the liver and largely exert metabolic functions,whereas non-parenchymal cells (NPCs)—comprising Kupffer cells, dendritic cells and liver sinu-soidal endothelial cells—control the immunological state within this organ. Here, we comparedthe suitability of two isolation methods for murine liver NPCs. Liver perfusion (LP) with collage-nase/DNase I applied via the portal vein leads to efficient liver digestion, whereas the modified liverdissociation (LD) method combines mechanical dissociation of the retrieved organ with enzymaticdegradation of the extracellular matrix. In cases of both LP and LD, NPCs were enriched by subse-quent gradient density centrifugation. Our results indicate that LP and LD are largely comparablewith regards to the yield, purity, and composition of liver NPCs. However, LD-enriched liver NPCsdisplayed a higher degree of activation after overnight cultivation, and accordingly were less respon-sive towards stimulation with toll-like receptor ligands that are frequently used as adjuvants, e.g., innano-vaccines. We conclude that LP is more suitable for obtaining liver NPCs for subsequent in vitrostudies, whereas LD as the less laborious method, is more convenient for parallel isolation of largernumbers of samples for ex vivo analysis.

Keywords: liver perfusion; liver dissociation; liver sinusoidal endothelial cells; Kupffer cells;dendritic cells; adjuvant; toll-like receptor ligand; liposome

1. Introduction

The liver is a central metabolic organ, but also plays an important immunologicalrole, serving to maintain tolerance under homeostatic conditions [1–3]. The liver is com-prised of hepatocytes, which make up approximately two thirds of its total cell populationand exert largely metabolic functions, comprising the synthesis of numerous types ofplasma proteins—for example, albumin [4], fatty acids [5], carbohydrates [6], and bile acidmetabolism [7]—but are also pivotal for detoxification [8]. The heterogeneous group ofnon-parenchymal cells (NPCs) plays an important role in immune regulation [9]. LiverNPCs comprise liver sinusoidal endothelial cells (LSECs, approximately 50% of NPCs),Kupffer cells (KCs, 20%) that largely constitute the resident macrophage population, anddendritic cells (DCs, 25%). The minor fraction of liver NPCs is mainly composed of innateimmune cells comprising natural killer (NK) cells and NKT cells [10], biliary epithelial cells [7],

Int. J. Mol. Sci. 2022, 23, 6543. https://doi.org/10.3390/ijms23126543 https://www.mdpi.com/journal/ijms

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and hepatic stellate cells (HSC) [11]. The latter are located in the perisinusoidal region be-tween the sinoids and hepatocytes, and under steady state conditions remain in a quiescentstate, but play an important role in the induction of liver diseases [12]. HSC are activated inresponse to liver damage and in this state constitute the main producers of extracellular matrixfactors—thereby promoting liver fibrosis [13]. Furthermore, chronic inflammation of a fibroticliver enhances the likeliness of hepatocellular carcinoma (HCC) development [14]. HCC inturn reprograms HSC to differentiate towards cancer-associated fibroblasts, which besidesother cell types such as tumor-associated macrophages, shape the immune-evasive tumormicroenvironment [15]. With regard to the major liver NPC fractions, KCs were recognizedearly on as promoting liver diseases [16] such as nonalcoholic fatty liver disease [17] andalcoholic hepatitis [18]. In general, KCs have been attributed protolerogenic functions [19,20].Similar to KCs, liver DCs have been reported to exert protolerogenic functions under steadystate conditions [21], but have also been reported to display a stimulated, proimmunogenicstate at later stages of NAFLD and to contribute to liver fibrosis [22].

LSECs form the lining of hepatic sinusoids and are characterized by numerous pore-like structures, termed fenestrae, which allow the rapid transport of, e.g., lipoproteins fromhepatocytes into the sinusoidal blood stream [23]. Furthermore, LSECs release vasodilatorymediators upon shear stress, and impaired generation of these factors has been implicated innon-alcoholic fatty liver disease [24]. Moreover, LSECs express numerous types of endocyticscavenger and C-type lectin receptors [25] as well as the Fcγ receptor IIb (CD32) [26], whichenable the uptake of different types of pathogens [27,28] and immune complexes [26] andpromote immune homeostasis by inducing regulatory T cells [29].

Altogether, liver NPCs on the one hand have the common property of maintainingantigen-specific T cell tolerance under homeostatic conditions [30,31], but on the other hand,may contribute to NAFLD and other liver diseases in cases of dysfunction [24,32,33]. LiverNPCs have also come into the focus of nanodrug-associated research as a large fractionof systemically applied nano-formulations accumulates in the liver [34–36]. Nanodrugshave the potential to co-deliver various agents in a tissue- and even cell type-specific manner,thereby reducing toxicity and other unwanted side effects associated with non-specific uptakeof conventional therapeutics [37]. Therefore, both with regard to nanodrugs designed forthe treatment of liver diseases [38] as well as nano-formulations developed to address otherorgans and cell types, it is highly important to address potential uptake by the liver.

In this regard, most studies have demonstrated endocytic uptake of nanodrugs byKCs [39–42]. Moreover, we and others have reported that DCs and LSECs may alsosignificantly contribute in this regard [36]. The overall T cell tolerance-promoting functionof liver NPCs may even be detrimental in cases of nano-vaccines developed to addressantigen-presenting cells such as DCs in secondary lymphoid organs aimed at inducingtumor antigen-specific immune responses [9]. Therefore, it is of general interest to study indetail the interaction of liver NPCs with biologicals in vitro to test and eventually optimizetheir cell-targeting properties for subsequent in vivo studies. Along this line, ex vivoanalysis of liver NPCs at the end of in vivo experiments enables the evaluation of in vivocell type-targeting behavior and payload-dependent functional effects.

So far, liver perfusion (LP) is the standard method for NPC enrichment but requireslaborious in situ pretreatment of the organ with collagenase and DNase I [43–46]. Hence,this approach limits the number of samples that can be processed. This issue may beespecially important when liver NPCs need to be isolated from a number of animals afterin vivo treatment for ex vivo analysis. Therefore, we compared the suitability of LP with exvivo liver dissociation (LD) using a commercially available kit that allows for the parallelprocessing of numerous samples. We showed that both liver NPC enrichment proceduresyielded similar results in terms of overall cell numbers, composition, and viability whenassessed directly after isolation. However, after overnight cultivation, NPCs enriched byLD displayed a higher state of activation and were less responsive towards stimulation withdifferent toll-like receptor (TLR) ligands. Altogether, our results show that both methodsare suitable for liver NPC isolation. LD proved less laborious than LP but might be more

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suitable in cases of direct ex vivo analysis of freshly isolated samples, whereas LP is themethod of choice when liver NPCs are to be subjected to functional studies in vitro.

2. Results2.1. LP and LD Yield Comparable Numbers of Enriched Viable NPCs

LP-mediated NPC enrichment starts with in situ perfusion of the liver via the portalvein with a collagenase/DNase I digestion mixture (Figure 1, left panel), whereas in casesof LD, only extra-corporal enzymatic tissue digestion of liver tissue is performed (Figure 1,right panel). Hence, LP is more demanding and laborious compared to LD. We modifiedthe LD protocol by performing subsequent density centrifugation to enrich NPCs in theinterphase (Figure 1, lower panel). This step is part of the LP method by default.

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Figure 1. Schematic overview of liver non-parenchymal cell (NPC) enrichment by liver perfusion (LP) and liver dissociation (LD) and subsequent experimental readout. Left panel: LP mice were (a) anesthetized with ketamine/Xylazine, (b) the vena portae was cannulated and collagenase A/DNase I-containing PBS was infused for in situ tissue digestion. (c) The liver was retrieved, cut into pieces and further digested with collagenase A/DNase I. Liver tissue pieces were mashed to obtain a single cell suspension (not shown). Right panel: LD mice were (d) killed by cervical dislocation, and (e) the retrieved liver was cut into pieces and digested in an enzyme cocktail of proprietary composition (Miltenyi Biotec), (f) followed by mechanical dissociation using a gentleMACS (Miltenyi Biotec). Lower left panel: Liver cell suspensions generated by either method were (g) subjected to Histodenz density gradient centrifugation, (h) resulting in enrichment of liver NPCs in the interphase that was subjected to subsequent experiments. Lower right panel: differentially enriched liver NPCs were characterized in a freshly isolated state and after overnight incubation w/o and in the presence of adjuvants with regard to viability, surface marker expression, and cytokine contents by flow cytom-etry. (created with BioRender.com; accessed on 1 June2022).

Figure 1. Schematic overview of liver non-parenchymal cell (NPC) enrichment by liver perfusion (LP)

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and liver dissociation (LD) and subsequent experimental readout. Left panel: LP mice were (a) anes-thetized with ketamine/Xylazine, (b) the vena portae was cannulated and collagenase A/DNaseI-containing PBS was infused for in situ tissue digestion. (c) The liver was retrieved, cut into piecesand further digested with collagenase A/DNase I. Liver tissue pieces were mashed to obtain a singlecell suspension (not shown). Right panel: LD mice were (d) killed by cervical dislocation, and (e) theretrieved liver was cut into pieces and digested in an enzyme cocktail of proprietary composition(Miltenyi Biotec), (f) followed by mechanical dissociation using a gentleMACS (Miltenyi Biotec).Lower left panel: Liver cell suspensions generated by either method were (g) subjected to Histodenzdensity gradient centrifugation, (h) resulting in enrichment of liver NPCs in the interphase that wassubjected to subsequent experiments. Lower right panel: differentially enriched liver NPCs werecharacterized in a freshly isolated state and after overnight incubation w/o and in the presenceof adjuvants with regard to viability, surface marker expression, and cytokine contents by flowcytometry. (created with BioRender.com; accessed on 1 June 2022).

Both liver NPC enrichment methods resulted in similar outcomes in terms of totalyield (Figure 2A), the frequency of enriched CD45+ liver NPCs (50–60%; Figure 2B) and thecomposition of the NPC population with regard to F4/80+ macrophages (MACs), CD11c+

DCs, and CD32b+ LSECs (Figure 2C). However, it is worth mentioning that after densitycentrifugation, cell suspensions obtained by LD contained more debris than observed whenperforming LP (not shown).

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Both liver NPC enrichment methods resulted in similar outcomes in terms of total yield (Figure 2A), the frequency of enriched CD45+ liver NPCs (50–60%; Figure 2B) and the composition of the NPC population with regard to F4/80+ macrophages (MACs), CD11c+ DCs, and CD32b+ LSECs (Figure 2C). However, it is worth mentioning that after density centrifugation, cell suspensions obtained by LD contained more debris than ob-served when performing LP (not shown).

Figure 2. LP and LD yield comparable numbers and composition of liver NPCs, but LD-enriched cells appear dead when using FVD. (A) Total cell yield obtained after density centrifugation (mean ± SEM, each n = 19). (B) Frequencies of CD45+ cells within the enriched NPC suspensions were as-sessed by flow cytometry (mean ± SEM, each n = 19). (C) Composition of liver NPCs (CD11c+ DCs, F4/80+ MACs, CD32b+ LSECs) within the CD45+ cell population (mean, each n = 19; the gating strat-egy is shown in Figure S1). Statistically significant differences versus (B) *LP (t-test) and (D) * be-tween groups (two-way ANOVA/Tukey test) are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Next, we investigated the viability of enriched liver NPC populations. To this end, we employed FVD, which binds intracellular primary amines of dead cells [47]. In contrast to other live/dead markers, FVD-treated samples can be fixated, which, in contrast to DNA-intercalating agents (see below), allows flow cytometric analysis at later time points.

Unexpectedly, LP-enriched MACs displayed rather strong interactions with the FVD, which suggested that their viability was about 42% only as compared to about 80% in the case of LD-enriched MACs (Figure 2D, left panel). After overnight incubation, MACs in-teracted with the FVD to a high extent, irrespective of the liver NPC enrichment method—suggesting viability rates below 40%. On the contrary, incubation of liver NPCs both di-rectly after isolation as well as after overnight cultivation with Sytox, which intercalates the genomic DNA of dead cells [48], in general delineated >75% of MACs as viable. Freshly isolated DCs (Figure 2D, middle panel) and LSECs (Figure 2D, right panel) dis-

Figure 2. LP and LD yield comparable numbers and composition of liver NPCs, but LD-enriched cellsappear dead when using FVD. (A) Total cell yield obtained after density centrifugation (mean ± SEM,each n = 19). (B) Frequencies of CD45+ cells within the enriched NPC suspensions were assessed byflow cytometry (mean ± SEM, each n = 19). (C) Composition of liver NPCs (CD11c+ DCs, F4/80+

MACs, CD32b+ LSECs) within the CD45+ cell population (mean, each n = 19; the gating strategyis shown in Figure S1). Statistically significant differences versus (B) *LP (t-test) and (D) * betweengroups (two-way ANOVA/Tukey test) are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

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Next, we investigated the viability of enriched liver NPC populations. To this end, weemployed FVD, which binds intracellular primary amines of dead cells [47]. In contrastto other live/dead markers, FVD-treated samples can be fixated, which, in contrast toDNA-intercalating agents (see below), allows flow cytometric analysis at later time points.

Unexpectedly, LP-enriched MACs displayed rather strong interactions with the FVD,which suggested that their viability was about 42% only as compared to about 80% inthe case of LD-enriched MACs (Figure 2D, left panel). After overnight incubation, MACsinteracted with the FVD to a high extent, irrespective of the liver NPC enrichment method—suggesting viability rates below 40%. On the contrary, incubation of liver NPCs bothdirectly after isolation as well as after overnight cultivation with Sytox, which intercalatesthe genomic DNA of dead cells [48], in general delineated >75% of MACs as viable. Freshlyisolated DCs (Figure 2D, middle panel) and LSECs (Figure 2D, right panel) displayedsimilar FVD/Sytox binding patterns, showing moderately lower viability when enrichedby LP and incubated with FVD, whereas no difference in viability was apparent in thecase of Sytox incubation. These method-dependent differences were less obvious afterovernight incubation, suggesting largely comparable viability rates determined by eitherviability dye. Similar results were obtained when using FVD conjugated with a distinctfluorochrome, and 7-AAD instead of Sytox (not shown).

Taken together, these observations suggest that LP-mediated enrichment of liver NPCsresulted in stronger interactions with the FVD than after LD enrichment (MACs > DCs, LSECs).

The unexpected finding of enhanced engagement of the FVD by MACs prompted usto perform a comparative viability analysis of spleen cells after isolation as well as afterovernight incubation using FVD and Sytox in parallel (Figure S2). None of the freshlyisolated splenic immune cell types showed stronger engagement of the FVD than of Sytox.However, after overnight culture B cells, MACs and PMNs displayed a lower viability ratewhen using FVD for assessment as compared to Sytox. Comparable findings were madewhen using FVD with a distinct fluorochrome and 7-AAD instead of Sytox (not shown).

Due to the cardinal advantage of FVD over Sytox/7-AAD to fixate samples aftertreatment, enabling flow cytometric measurements at later time points, we also testedwhether Annexin V—which detects phosphatidyl serin on the surface of early/late apop-totic cells [49] and allows for sample fixation as well—could be used instead of FVD.However, a considerable proportion of LD-enriched liver NPCs engaged Annexin V to asimilar extent as that observed for FVD (Figure S3).

Altogether, these observations suggest that DNA-intercalating agents (Sytox, 7-AAD)are more reliable for the viability analysis of liver NPCs.

2.2. Liver NPCs Enriched by LD Express Costimulatory Receptors at Higher Levels and ArePartially Refractory towards Stimulation

Our findings on the differential binding of liver NPCs to FVD viability dyes suggestedthat both liver NPC enrichment procedures may also affect their overall state of activation.Overnight-cultivated liver NPC populations displayed no method-associated differencesin expression of the antigen-presenting receptor MHCII (Figure 3, Ctrl groups). In contrast,MACs, DCs and LSECs expressed the costimulatory receptors CD80 and CD86 to a higherextent when enriched by LD as compared to LP.

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Figure 3. LD-enriched liver NPCs are pre-activated, as deduced from elevated activation marker expression. Liver NPCs enriched by LP and LD were seeded into wells of 96 well plates (105/100µL) and stimulated overnight with the agents indicated or left untreated. The man fluorescence intensity (MFI) of surface markers was assessed by flow cytometry (mean ± SEM, n = 4). Statistically signifi-cant differences versus *according Ctrl and versus +LP are indicated (one way ANOVA, Tukey test). *, + p < 0.05, **, ++ p < 0.01, ***, +++ p < 0.001.

To assess the suitability of LP- and LD-derived liver NPCs for in vitro testing of ad-juvants, e.g., as a component of nano-vaccines, we applied several TLR ligands—includ-ing the TLR4 ligand LPS, the TLR7/8 agonist R848, native mRNA (TLR3 agonist), the well-established liposomal carrier DOTAP, and DOTAP/mRNA complexes. MHCII expression of MACs enriched by either of the methods displayed was not altered by either stimulus. Similar observations were made for the co-stimulator CD80. In contrast, CD86 expression was upregulated in the case of LP-enriched MACs in response to either stimulus, whereas LD-derived MACs remained unresponsive in this regard. LP-enriched DCs upregulated MHCII and showed similar tendencies regarding both co-stimulators in response to stim-ulation, whereas DCs pre-activated due to enrichment by LD were refractory.

LP-enriched LSECs showed increased MHCII expression in response to LPS, R848 and DOTAP, but LD-enriched LSECs displayed only very moderately elevated levels. Similar to MACs and DCs, LD-derived LSECs in general scarcely upregulated either co-stimulator in response to stimulation. In contrast, LP-derived LSECs showed somewhat increased CD80 expression when stimulated with R848 and significant upregulation of CD86 upon application of most stimuli.

Figure 3. LD-enriched liver NPCs are pre-activated, as deduced from elevated activation markerexpression. Liver NPCs enriched by LP and LD were seeded into wells of 96 well plates (105/100 µL)and stimulated overnight with the agents indicated or left untreated. The man fluorescence intensity(MFI) of surface markers was assessed by flow cytometry (mean ± SEM, n = 4). Statistically significantdifferences versus * according Ctrl and versus +LP are indicated (one way ANOVA, Tukey test).*, + p < 0.05, **, ++ p < 0.01, ***, +++ p < 0.001.

To assess the suitability of LP- and LD-derived liver NPCs for in vitro testing of adju-vants, e.g., as a component of nano-vaccines, we applied several TLR ligands—includingthe TLR4 ligand LPS, the TLR7/8 agonist R848, native mRNA (TLR3 agonist), the well-established liposomal carrier DOTAP, and DOTAP/mRNA complexes. MHCII expressionof MACs enriched by either of the methods displayed was not altered by either stimulus.Similar observations were made for the co-stimulator CD80. In contrast, CD86 expres-sion was upregulated in the case of LP-enriched MACs in response to either stimulus,whereas LD-derived MACs remained unresponsive in this regard. LP-enriched DCs upreg-ulated MHCII and showed similar tendencies regarding both co-stimulators in response tostimulation, whereas DCs pre-activated due to enrichment by LD were refractory.

LP-enriched LSECs showed increased MHCII expression in response to LPS, R848 andDOTAP, but LD-enriched LSECs displayed only very moderately elevated levels. Similarto MACs and DCs, LD-derived LSECs in general scarcely upregulated either co-stimulatorin response to stimulation. In contrast, LP-derived LSECs showed somewhat increasedCD80 expression when stimulated with R848 and significant upregulation of CD86 uponapplication of most stimuli.

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In line with the partially activated state of LD-enriched liver NPC populations afterovernight cultivation, the respective cell culture supernatants contained higher concentra-tions of TNF-α, IL-6, and IL-10 than in the case of LP-derived NPCs (Figure 4). Type I (IFN-α,-β) and type II (IFN-γ) interferons were scarcely detectable under basal conditions. Most cy-tokines, except for IFN-α, were upregulated to the highest extent by stimulation with R848and to a similar extent, in an enrichment method-independent manner. Only IL-10 showedmarkedly higher absolute levels in the case of LD-enriched NPCs than those observed forLP-derived NPCs. Stimulation with LPS also increased cytokine concentrations—againexcept for IFN-α—albeit to a lower extent than that induced by R848. Again, for some cy-tokines (TNF-α, IL-6, IL-10), levels were somewhat higher in the case of LD-derived NPCs.Only in the case of IFN-γ did LP-derived NPCs produce more cytokine than that observedfor LD-enriched NPCs. mRNA and DOTAP, when applied separately, exerted no stimula-tory effect on the production of either cytokine—except for IFN-γ, which was markedlyupregulated in the case of LP-enriched NPCs treated with mRNA. DOTAP/mRNA com-plexes, however, specifically increased type I IFN concentrations—again, interestingly, to ahigher extent in the case of LP- than LD-derived liver NPCs.

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In line with the partially activated state of LD-enriched liver NPC populations after overnight cultivation, the respective cell culture supernatants contained higher concen-trations of TNF-, IL-6, and IL-10 than in the case of LP-derived NPCs (Figure 4). Type I (IFN-, -) and type II (IFN-) interferons were scarcely detectable under basal conditions. Most cytokines, except for IFN-, were upregulated to the highest extent by stimulation with R848 and to a similar extent in an enrichment method-independent manner. Only IL-10 showed markedly higher absolute levels in the case of LD-enriched NPCs than those observed for LP-derived NPCs. Stimulation with LPS also increased cytokine concentra-tions—again except for IFN-—albeit to a lower extent than that induced by R848. Again, for some cytokines (TNF-, IL-6, IL-10), levels were somewhat higher in the case of LD-derived NPCs. Only in the case of IFN- did LP-derived NPCs produce more cytokine than that observed for LD-enriched NPCs. mRNA and DOTAP, when applied separately, exerted no stimulatory effect on the production of either cytokine—except for IFN-, which was markedly upregulated in the case of LP-enriched NPCs treated with mRNA. DOTAP/mRNA complexes, however, specifically increased type I IFN concentrations—again, interestingly, to a higher extent in the case of LP- than LD-derived liver NPCs.

Figure 4. Liver NPCs generate cytokines in an adjuvant-specific manner and depending on the en-richment method used. Supernatants of differentially stimulated liver NPCs (see Figure 3) were as-sayed for cytokine contents in culture supernatants (mean ± SEM, n = 3). Statistically significant differences versus * according Ctrl and versus +LP are indicated (one way ANOVA, Tukey test). **, ++ p < 0.01, ***, +++ p < 0.001.

Altogether, these results suggest that LD results in the partial activation of enriched liver NPCs, which in the case of surface activation markers prevented stimulation-de-pendent upregulation. In agreement, LD-enriched liver NPCs showed higher basal levels of some cytokines, but in contrast to inducing non-responsiveness on the level of surface markers, were capable of upregulating cytokine production in response to stimulation.

3. Discussion The immunological role of liver NPCs, and the frequent observation that nanodrugs

intended to address other cell types are frequently enriched in this organ, underline the importance of studying the interactions of liver NPC populations with nanodrugs and the functional consequences thereof. Furthermore, liver NPCs have also been recognized as

Figure 4. Liver NPCs generate cytokines in an adjuvant-specific manner and depending on theenrichment method used. Supernatants of differentially stimulated liver NPCs (see Figure 3) wereassayed for cytokine contents in culture supernatants (mean ± SEM, n = 3). Statistically significantdifferences versus * according Ctrl and versus +LP are indicated (one way ANOVA, Tukey test).**, ++ p < 0.01, ***, +++ p < 0.001.

Altogether, these results suggest that LD results in the partial activation of enrichedliver NPCs, which in the case of surface activation markers prevented stimulation-dependentupregulation. In agreement, LD-enriched liver NPCs showed higher basal levels of somecytokines, but in contrast to inducing non-responsiveness on the level of surface markers,were capable of upregulating cytokine production in response to stimulation.

3. Discussion

The immunological role of liver NPCs, and the frequent observation that nanodrugsintended to address other cell types are frequently enriched in this organ, underline theimportance of studying the interactions of liver NPC populations with nanodrugs and thefunctional consequences thereof. Furthermore, liver NPCs have also been recognized as an

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interesting target population for nanodrug-mediated approaches aimed at exploiting theirintrinsic protolerogenic potential to inhibit unwanted immune reactions, as in the case ofallergies [50]. Moreover, in the case of anti-tumor nano-vaccination approaches, it may bean important issue to overcome their tolerance-promoting properties by applying suitableadjuvants, as assessed in this study [9].

Therefore, the suitability of liver NPC isolation methods is an important issue. In thisregard, LP has been well established over the last decades, and numerous modificationshave been tested—as summarized in Table 1. However, LP requires time-consuming insitu treatment of the liver, which may limit the number of samples that can be processed,e.g., for ex vivo analysis after in vivo treatments—as for example in the case of biodistribu-tion studies of nano-carriers [36]. Therefore, we compared LP with LD as an alternativeapproach in this study.

Table 1. Protocols for liver NPC enrichment.

Liver Digestion Access to theLiver Species NPC Yield (×106) Gradient

Centrifugation Viability (%) 1 Reference

Perfusion(aeration apparatus) Vp r - - - [51]

0.05% Collagenase/0.10%hyaluronidase Vp r - - - [52]

Pronase (Streptomyces griseus) Vp r 2–15 yes - [53]

Pronase (Streptomyces griseus) - r 14/gr - - [54]

Collagenase - r 0.5–0.6/gr Percoll - [55]

Pronase/DNAse/Collagenase Vp r 26 (LSEC), 13 (KC) Stractan 90 [56]

Collagenase Vp m 9 Metrizamide - [57]

Collagenase/DNAse Vp m - Histodenz - [43]

Collagenase/DNAse Vp m - Histodenz - [44]

Collagenase IV/Pronase Vp m - Percoll 97 [9]

Collagenase IV(C. histolyticum) ex vivo h KC: 1.8,

LSEC: 4.3/gr yes - [58]

EGTA/collagenase P ex vivo h 1.9 (KC), 0.27(LSEC)/gr >90 [59]

According to [53] Vp m - yes - [60]

Collagenase II ex vivo m 5/gr tissue - - [61]

Collagenase I Vp r KC: 3–5,LSEC: 18–20 Percoll - [62]

EGTA/collagenase P(two-step) ex vivo h - Yes - [63]

Collagenase/DNAse Vp m 3.33 Histodenz - [36]

h: human, KC: Kupffer cell; LSEC: liver sinusoid endothelial cell; m: murin, Vp: vena portae. 1 numbers givenwhen viability was assessed in the study.

We show that both enrichment methods yielded overall comparable results in termsof yield of CD45+ liver NPCs [9], displaying similar compositions of NPC populationswith regard to DCs, MACs, and LSECs. Surprisingly, however, we observed that LD-enriched liver NPCs, and MACs especially, showed unspecific interactions with the FVD,and thereby would be considered dead. Similar findings were also made for the other liverNPCs populations. Furthermore, both LP- and LD-enriched MACs displayed low viabilityafter overnight cultivation when using FVD. In contrast, DNA-intercalating agents (Sytox,7-AAD) identified only <25% of MACs as dead, even after overnight incubation. Besidesthis, splenic MACs and PMN were also characterized by stronger interaction with FVDthan DNA-intercalating dyes after overnight cultivation. Therefore, these findings suggestthat endocytically active cell types in a viable state are prone to internalize FVD, which

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favors the use of DNA-binding agents for delineating viability. It is unclear as of yet whichreceptors may play a role in this regard.

However, LD enrichment also resulted in partial activation of the assessed liver NPCtypes, which may compromise the outcome of in vitro studies aimed, e.g., at assessing thestimulatory activity of agents—as exemplified in this study for several TLR ligands. Inthis regard, the responsiveness of LD-dependently pre-activated liver NPCs on the levelof surface markers was strongly impaired. LD-enriched NPCs generated easily detectableamounts of TNF-α, IL-6, and IL-10—even in the absence of stimulatory agents—but thedifference towards LP-derived NPCs was most apparent in the case of IL-10. This potentanti-inflammatory cytokine has been implicated as an effector molecule upregulated inresponse to different types of stress [64] and has been shown to contribute in an essentialmanner to the overall pro-tolerogenic role of liver NPCs [65]. KCs have been reported toexpress IL-10 receptor at high levels, resulting in sustained activation of the transcriptionfactor signal transducer and activator of transcription (STAT)3 [66]. STAT3 in turn is amajor driver of IL-10 expression, thereby establishing a positive feedback loop for thiscytokine [67]. However, LD-derived liver NPCs were still largely responsive towardsstimulation on the cytokine level. Here, we observed that R848, and to lesser extent LPS,was a potent stimulator of TNF-α, IL-6, IL-10, and IFN-γ production—as previously shownby us and others [68]. In addition, we also demonstrated that complexed mRNA yieldedconsiderable induction of type I IFN, as has also been described for antigen-presenting cellsin secondary lymphoid organs upon transfection with mRNA-delivering liposomes [69–73].

Our results show that LP-derived liver NPCs can be used to study, e.g., the stimulatoryeffects of adjuvant-loaded nanodrugs in terms of surface activation marker expression andcytokine production. With regards to the latter, it will also be interesting to assess cytokineproduction on the single cell level by intracellular cytokine detection [74]. In the case offluorescence-labeled nano-carriers, it will also be possible to study their liver NPC type-specific engagement—especially in the case of nanodrugs decorated with, e.g., C-type lectinreceptor-targeting moieties such as carbohydrates [75]. Finally, in the case of nano-vaccinesthat co-deliver an antigen, subsequent cocultures of accordingly pretreated liver NPCswith antigen-specific T cells will show whether adjuvant-mediated NPC activation is ableto overcome their intrinsic protolerogenic properties and result in efficient activation ofantigen-specific T cells, e.g., to induce profound anti-tumor T cell responses [76].

Altogether, we conclude that LP is more reliable than LD for obtaining liver NPCs forsubsequent in vitro studies. However, LP represents a laborious technique and well-trainedoperators are needed (Table 2). Therefore, LD is the method of choice in the case liver NPCsthat need to be derived from a larger number of in vivo-treated animals for subsequentex vivo analysis. In this case, the potential of LD to process a larger number of samplesin parallel enables the completion of NPC isolation within a shorter period of time ascompared to the rather continuous mode of handling in the case of LP. Additionally, ourfinding that LD partially activates NPCs in the course of subsequent overnight culture isnot of primary concern when samples are to be analyzed directly after NPC isolation.

Table 2. Advantages and disadvantages of using LP or LD for NPC enrichment.

Aspect LP LD

Total yield +++ +++Purity (CD45+) 62% 52%NPC viability 1 MAC 89% 2, 76% 3 82%, 89%

DC 86%, 62% 81%, 72%LSEC 92%, 82% 90%, 91%

NPC activation state o/+ +++NPC responsiveness towards stimulation ++ o/+Preparation time 4–5 h 2–3 hCosts + +++

1 given for Sytox; 2 freshly isolated; 3 after overnight culture. O, unaltered; +, low; ++, intermediate, +++, high.

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4. Materials and Methods4.1. Animals

C57BL/6 mice were bred and housed in the Translational Animal Research Center(TARC) of the Johannes Gutenberg-University Mainz under specific pathogen-free condi-tions on a standard diet, according to the guidelines of the regional animal care committee.The guide for the care and use of laboratory animals [77] as well as the 3R principlesin laboratory animal experiments [78] were followed. Mice were sacrificed at an age ofbetween 12–16 weeks for organ retrieval according to § 4(3) TierSchG.

4.2. Spleen Cells

Spleens were mashed using a sterile syringe plunger on a PBS pre-soaked 40 µmcell strainer (EASYstrainer™; Sarstedt, Nümbrecht, Germany). Mashed spleen tissue waswashed through the strainer using 10 mL of PBS/2 mM EDTA buffer, and cells werepelleted by centrifugation (1200 rpm, 10 min, 4 ◦C).

4.3. Enrichment of Liver NPCs

Mice NPCs were enriched by liver perfusion (LP) and liver dissociation (LD). For LP,mice were anesthetized with a Ketamine/Xylazine mixture (Ketamine 120 mg/Kg; Xylasin16 mg/kg) and the abdominal cavity was opened (see Figure 1, left panel). The venaportae was cannulated and flushed with 20 mL of HBSS (Hank’s Balanced Salt Solution;ThermoFisher, Waltham, MA, USA) containing 100 U/mL of collagenase A (Sigma-Aldrich,St. Louis, MO, USA) and 10 µg/mL of DNase I (Sigma-Aldrich, St. Louis, MO, USA).Then, the liver was retrieved, cut into pieces, and incubated for 15 min at 37 ◦C in a50 mL tube with PBS containing 100 U/mL of collagenase A and 10 µg/mL of DNase I.Afterwards, the liver tissue was mashed through a 70 µm nylon cell strainer and medium(DMEM (Dulbecco’s Modified Eagle Medium)/F-12 GlutaMAX™, ThermoFisher Scientific)was applied. The cell suspension was subjected to density centrifugation using 30%Histodenz-HBSS (both from Sigma-Aldrich, St. Louis, MO, USA) gradient centrifugationas described [45] (see Figure 1, lower panel).

For LD-mediated NPC enrichment, mice were killed by cervical dislocation and theliver was retrieved (see Figure 1, right panel). An enzyme-dependent dissociation mixtureof proprietary composition (Liver dissociation kit; Miltenyi Biotec, Bergisch-Gladbach,Germany) was preincubated for 15 min in C tubes (Miltenyi Biotec). Then, liver tissueswere cut in little pieces and transferred into prepared C tubes. The latter were placed intoa gentleMACS Dissociator and the tissue was minced (two times program m_liver_03).Afterwards, the cell suspension was incubated for 30 min at 37 ◦C under continuousshaking, followed by another round of gentleMACS-mediated disintegration (two timesprogram m_liver_04). The samples were cleared using a Cell Strainer (100 µm; Sarstedt)and the liver NPCs were enriched by density centrifugation (see above).

4.4. Stimulation of Liver NPCs

Liver NPCs were seeded into wells of 96-well plates (105/100 µL) and were treatedovernight with LPS, (100 ng/m; Kenilworth, NJ, USA), R848 (1 µg/m, Ínvivogen, San Diego,CA, USA), ovalbumine-encoding mRNA (1 µg/mL; Tri-Link, San Diego, CA, USA), DOTAP(6.4 µL/mL; Roth, Weiden, Germany), and DOTAP/mRNA complexes, which were formedby mixing both components at the mentioned amounts as recommended by the manufac-turer (Roth).

4.5. Flow Cytometry

Single cell suspensions were washed (2% FCS in PBS), and Fc receptors were blockedwith aCD16/CD32 (clone 2.4G2 clone; Biolegend, San Diego, CA, USA) for 10 min. Af-terwards, cells were incubated with receptor-specific antibodies (Table S1) obtained fromThermoFisher (Waltham, MA) or BD Biosciences (Franklin Lakes, NJ, USA) for 20 minat 4 ◦C. Afterwards, cell viability was assessed by using fixable viability dye (FVD)-450

Int. J. Mol. Sci. 2022, 23, 6543 11 of 14

and -780, 7AAD, Sytox AADvanced (all from ThermoFisher) and Annexin V-AF647 (Biole-gend), as recommended by the manufacturer. Samples were subjected to flow cytometryusing a Attune NxT Flow Cytometer (Thermo Scientific) and an LSRII (BD Biosciences),and analyzed with Attune NxT ((ThermoScientific and FlowJo software, respectively (BDBiosciences)). The gating strategies for liver NPCs (see Figure S1) and splenic leukocytes(see Figure S2A) are depicted in the supplementals.

4.6. Cytokines

Supernatants of cell cultures were retrieved and stored at −20 ◦C for subsequentanalysis. TNF-α, IFN-γ, IL-6, IL-1ß, and IL-10 were quantified using a Cytometric BeadAssay (CBA; BD Biosciences) as recommended by the manufacturer. Results were analyzedusing FCAP Array Analysis Software v.1.0.1 (BD Biosciences). IFN-α and IFN-β weremeasured using Legendplex reagents as recommended and data were analyzed usingQognit Legendplex Analysis Software (both from Biolegend).

4.7. Data Analysis

Data were analyzed using GraphPad PRISM v5 and v9 software (GraphPad Software Inc.,San Diego, CA, USA). Data are given as mean ± SEM for the indicated number of inde-pendent experiments. Statistical differences were determined by unpaired Student’s t-testswhen comparing two groups, one way ANOVA followed by post-hoc Tukey‘s test whencomparing multiple groups monitored at a given time point, and two-way ANOVA/Tukeytests when comparing multiple groups assessed at different time points—assuming signifi-cant differences at p < 0.05 in either case.

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23126543/s1.

Author Contributions: Conceptualization, C.M.-M., M.L.C. and M.B.; methodology, C.M.-M. andM.L.C.; software, C.M.-M., M.L.C., M.S., M.J.L., Y.Z., J.E.C.-G. and N.R.; validation, C.M.-M., M.L.C.,M.S., M.J.L., Y.Z., J.E.C.-G., N.R. and M.B.; formal analysis, C.M.-M., M.L.C., M.S., M.J.L., Y.Z.,J.E.C.-G. and N.R.; investigation, C.M.-M., M.L.C., M.S., M.J.L., Y.Z., J.E.C.-G. and N.R.; resources,C.M.-M., M.L.C., M.S., M.J.L., Y.Z., J.E.C.-G. and N.R.; data curation, C.M.-M., M.L.C., Y.Z. andM.B.; writing—original draft preparation, C.M.-M., M.L.C. and M.B.; writing—review and editing,C.M.-M., M.L.C., S.G. (Stephan Grabbe), S.G. (Stephan Gehring) and M.B.; visualization, C.M.-M.,M.L.C., Y.Z. and M.B.; supervision, M.L.C., S.G. (Stephan Gehring) and M.B.; project administration,S.G. (Stephan Grabbe), S.G. (Stephan Gehring) and M.B.; funding acquisition, S.G. (Stephan Grabbe),S.G. (Stephan Gehring) and M.B. All authors have read and agreed to the published version ofthe manuscript.

Funding: This research was funded by Deutsche Forschungsgemeinschaft (SFB1066), grant numbersB4 (S.G. (Stephan Grabbe)) and B15 (S.G. (Stephan Gehring), M.B. (Matthias Bros)).

Institutional Review Board Statement: Mice were sacrificed for organ retrieval according to § 4(3)TierSchG.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Acknowledgments: We thank the Research Center for Immunotherapy (FZI; University MedicalCenter, Mainz, Germany) for their continuous support.

Conflicts of Interest: The authors declare no conflict of interest.

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